PSC BIOINFORMATICS TRAINING LED TO THE FULL SEQUENCING OF A LITTLE
UNDERSTOOD BACTERIOPHAGE

Like a mosquito on a summer evening, a bacteriophage
is either feasting or in search of its next meal. But a bacteriophage
isn’t interested in human blood and isn’t flying around your backyard.
Bacteriophages — aka phages — are microscopic killers of
bacteria. Wherever you find bacteria, which is nearly everywhere,
you’ll find phages.

A phage is a virus, and the feasting begins when, like any virus, it
attaches to its bacterium host and injects its DNA. The phage DNA
hijacks the bacterium’s machinery and begins to reproduce itself.
Soon, the bacterium is teeming with new phages that burst forth
from the bacterium, destroying it. The hunt for a new victim begins.

Not long after Canadian scientist Felix
d’Herelle gave a name to viruses that infect bacteria in 1917, he
recognized their potential to treat disease. Using sewage, he isolated
the dysentery phage and put it in solution. After he and other doctors
in a Paris hospital drank a few pints to test it, they administered
their phage solution to children dying from dysentery, who were cured
the next day.

D’Herelle traveled across Europe and the Soviet Union with his
microscopic miracles. When Alexander Fleming stumbled on
penicillin in 1928, however, the world had a magic bullet for
bacterial infections. Except in a few countries, the use of phages
declined into oblivion.

Aleisha Dobbins, Howard University.

Today, with fast evolving, antibiotic-resistant bacteria, phages are
back in the spotlight. With bioinformatics tools, researchers are
seeking to understand them at the level of DNA and genes.

Populous Parasites

With phages, the sheer numbers are almost scary — 10 million
populate a milliliter of seawater, about 50 drops. Phages comprise
the majority of organisms on the planet, and through the recycling
of carbon in the oceans may be responsible for up to a quarter of
the planet’s energy turnover.

Electron micrograph of two SP6 virus particles, the roughly
hexagonal-shaped head is about 50 nanometers in diameter.

“When people hear the word ‘virus,’ they think trouble,” says
Dobbins. “But phages kill bacteria and have no effect on humans.
They can be used in addition to antibiotics. With interest in phage
therapy resurfacing, it’s important to do sequence analysis and map
the genes of more phages.”

Research in phages is also part of the effort to defeat human viral
disease. Having all the genes they need to replicate themselves,
phages are similar to viruses that invade humans, but easier to study
because their hosts are bacteria, not humans. If researchers learn
how phages assemble their protein houses, called capsids, they may
develop the means to dismantle them. Without the capsid shells,
both phages and human viruses are harmless bits of floating DNA.

An electron microscope image of the Salmonella bacterium.
(Courtesy of Rocky Mountain Laboratories,NIAID,NIH)

The SP6 phage in particular attracts attention because of its host.
Phages are picky parasites. Each one invades a particular bacterium,
and SP6 goes after Salmonella, the nasty bacteria that dwell in raw
meat and cause food poisoning. While SP6 hasn’t been yet been used
to treat food poisoning, it is widely used in biotechnology.

SP6’s RNA polymerase, an enzyme that transcribes DNA into RNA, is
commonly used in genetic technology to modify and clone the DNA
sequences of bacteria. Despite wide use, SP6 had not been
sequenced and most of its genes had not been identified before
Dobbins’ work.

PHAGE RESEARCH MAY HELP TO DEFEAT HUMAN VIRAL DISEASE

Getting Answers

As a Ph.D student working on her dissertation, Dobbins planned to
focus on one of SP6’s genes. Her plans became more ambitious,
however, when she went to a PSC bioinformatics workshop, led by
PSC scientist and sequence-analysis expert Hugh Nicholas. Through
the workshop, Dobbins gained the ability to tackle the much larger
project of the entire SP6 genome.

“Through this workshop,” says Dobbins, “I gained knowledge of the
bioinformatics tools I needed to sequence the genome. And I learned
how to use software to identify the termination sequences.”

From July 12 to 23, 2004, PSC hosted 19 faculty and staff
from nine universities for its two-week course,
“Developing Bioinformatics Programs.” PSC
scientists Hugh Nicholas (1st row center) and David
Deerfield (2nd row right) led the course. Five interns from
three universities stayed at PSC for five weeks to continue
work on their research projects.

Tools for the Job

The tools of bioinformatics, which marry information
science and statistics with the life sciences, allow
researchers to understand biological systems like never
before. But researchers need to learn about these new and
rapidly improving tools. PSC’s “Developing Bioinformatics
Programs” course introduces faculty and graduate students
from minority-serving institutions to the computational,
mathematical, and biological issues of bioinformatics.

“Bioinformatics computer programs in general involve
implementing a mathematical model and comparing the model
with the data to see how they relate to each other,”
says PSC scientist Hugh Nicholas. “The most common
bioinformatics task involves taking a sequence from a
biologist’s laboratory and comparing it to all sequences in
the database looking for relationships according to a
mathematical model of sequence evolution.”

The two-week workshop trains faculty who plan to establish
an introductory bioinformatics course at their home
institution, and graduate students, such as Aleisha
Dobbins, to use bioinformatics tools to complete a research
project. The course is sponsored by a grant from the
Minority Access to Research Careers Branch of the Branch of
Division of Minority Opportunity in Research of the
National Institute of General Medical Sciences. It grew out
of a bioinformatics workshop originally developed through
support from NIH’s National Center for Research Resources,
which also supports Tourney, PSC’s sequence-analysis
computer, used during the workshop and by university
students in courses developed through the workshop. Tourney
is available to support bioinformatics course work at any
U.S. academic institution.

Nicholas introduced Dobbins to researchers at the Pittsburgh
Bacteriophage Institute (PBI) at the University of Pittsburgh. Dobbins
and PBI co-directors Roger Hendrix and Graham Hatfull decided that
rather than examining one gene, it made sense to sequence and
examine the entire genome. This would allow them to compare SP6
with other well-known phages and, perhaps, to draw conclusions
about SP6’s evolution, information that relates directly to the ability
of bacteria to evolve and defeat antibiotics.

Phages and bacteria evolve in conjunction, bound together in the
race to outwit one another and survive. Through this evolutionary
drama, phages introduce new genes into the bacteria population.
“Most human pathogens are as toxic as they are because of genes
that were brought in by phages,” says PBI’s Hendrix. “There’s a lot of
interest in what this population looks like and by comparing them to
each other we can start to see how the population evolved up to
where it is now.”

At PBI, Dobbins sequenced SP6’s entire genome of over 40,000
nucleotides, the building blocks of DNA. She also identified some of
the genes and their order. With training from Nicholas and the PSC
workshop, she used Tourney, PSC’s sequence-analysis computer, to
identify the terminator sequences — regions of the genome that
signal RNA polymerases to stop transcribing and disconnect. With
Tourney, Dobbins also compared SP6 sequences with databases of
known phage gene sequences and thereby identified SP6’s genes.

Dobbins identified SP6 as being part of the T7 phage family, which
includes T7 and T3, two of the most well researched phages — a
family relation that had been suspected, but not verified. Because of
their similarities, Dobbins used a template of the T7 RNA
polymerase, which is also used in genetic technology, to build a
model of SP6’s polymerase, the gene she hoped to examine in her
original research plan.

Phages in the T7 family presumably evolved from the same ancestor
as SP6, and have many similarities in sequence and gene placement.
But there are many family mysteries. Through comparative analysis,
Dobbins found that one sequence of genes appearing in the same
place in most phages in the T7 family was in a much different place
in SP6. The group is not only in a different place, but reversed in
order. As with any research, answers spark new questions.

Dobbins’ work will feed an ongoing discussion about phage
evolution. While some believe that they evolved from a common
ancestor millions of years ago, others argue that similar structures
arose independently, or diverged more recently.

“The evolution of bacteriophages has not totally been traced,” said
Dobbins. “We don’t know how they have evolved or what kind of
effect this phage had on bacteria. We completed the sequence and
found that it had 52 genes, and of the 52, 64 percent are unique to
SP6. A lot of additional work needs to be done to identify the
function of those genes.”